Glucagon analogs exhibiting physiological solubility and stability

ABSTRACT

Modified glucagon peptides are disclosed having improved solubility and stability, wherein the native glucagon peptide has been modified by pegylation, or the addition of a carboxy terminal peptide selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or both.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national counterpart application ofinternational application serial No. PCT/US2006/043334 filed Nov. 6,2006, which claims priority to U.S. Provisional Patent Application No.60/734,307 filed Nov. 7, 2005. The entire disclosures ofPCT/US2006/043334 and U.S. Ser. No. 60/734,307 are hereby incorporatedby reference.

BACKGROUND

Hypoglycemia occurs when blood glucose levels drops too low to provideenough energy for the body's activities. In adults or children olderthan 10 years, hypoglycemia is uncommon except as a side effect ofdiabetes treatment, but it can result from other medications ordiseases, hormone or enzyme deficiencies, or tumors. When blood glucosebegins to fall, glucagon, a hormone produced by the pancreas, signalsthe liver to break down glycogen and release glucose, causing bloodglucose levels to rise toward a normal level. However for diabetics,this glucagon response to hypoglycemia may be impaired, making it harderfor glucose levels to return to the normal range.

Hypoglycemia is a life threatening event that requires immediate medicalattention. The administration of glucagon is an established medicationfor treating acute hypoglycemia and it can restore normal levels ofglucose within minutes of administration. When glucagon is used in theacute medical treatment of hypoglycemia, a crystalline form of glucagonis solubilized with a dilute acid buffer and the solution is injectedintramuscularly. While this treatment is effective, the methodology iscumbersome and dangerous for someone that is semi-conscious.Accordingly, there is a need for a glucagon analog that maintains thebiological performance of the parent molecule but is sufficientlysoluble and stable, under relevant physiological conditions, that it canbe pre-formulated as a solution, ready for injection.

Additionally, diabetics are encouraged to maintain near normal bloodglucose levels to delay or prevent microvascular complications.Achievement of this goal usually requires intensive insulin therapy. Instriving to achieve this goal, physicians have encountered a substantialincrease in the frequency and severity of hypoglycemia in their diabeticpatients. Accordingly, improved pharmaceuticals and methodologies areneeded for treating diabetes that are less likely to induce hypoglycemiathan current insulin therapies.

As described herein, high potency glucagon agonists are provided thatexhibit enhanced biophysical stability and aqueous solubility. Thesecompounds can be used in accordance with one embodiment to preparepre-formulated solutions ready for injection to treat hypoglycemia.Alternatively, the glucagon agonists can be co-administered with insulinto buffer the effects of insulin to allow for a more stable maintenanceof blood glucose levels. In addition, other beneficial uses ofcompositions comprising the modified glucagon peptides disclosed hereinare described in detail below.

SUMMARY

In accordance with one embodiment, analogs of glucagon are provided thathave improved solubility and stability as well as similar bioactivies,including similar or higher potency and selectivity at the glucagon andGLP-1 receptors, relative to the native glucagon peptide. In oneembodiment the glucagon analogs have at least 75% activity, or at least85% activity as native glucagon. In one embodiment, the glucagon analogsof the present invention have potency greater than glucagon.

In accordance with one embodiment a glucagon agonist is providedcomprising a glucagon peptide of SEQ ID NO: 45 or glucagon agonistderivative of SEQ ID NO: 45, wherein the side chain of an amino acidresidue at position 21 or 24 of said glucagon peptide further comprisesa hydrophilic moiety covalently bound to the amino acid residue. Inaccordance with one embodiment a glucagon agonist is provided comprisinga glucagon peptide selected from the group consisting of SEQ ID NO: 2,SEQ ID NO: 3, and SEQ ID NO: 4, and glucagon agonist derivatives of SEQID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, wherein the side chain of anamino acid residue at position 21 or 24 of said glucagon peptide furthercomprises a hydrophilic moiety covalently bound to the amino acidresidue. The present invention further encompasses pharmaceuticallyacceptable salts of said glucagon agonists. In accordance with oneembodiment the glucagon peptide is selected from the group consisting ofSEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, andthe hydrophilic moiety is a polyethylene glycol chain, having amolecular weight selected from the range of about 500 to about 40,000Daltons. In one embodiment the polyethylene glycol chain has a molecularweight selected from the range of about 500 to about 5,000 Daltons. Inanother embodiment the polyethylene glycol chain has a molecular weightof at least about 20,000 Daltons.

In another embodiment a glucagon agonist is provided comprising aglucagon peptide and a polyethylene glycol chain, wherein thepolyethylene glycol chain is covalently bound to residue 16, 17, 20, 21,24 or 29 of the glucagon peptide. The present invention also encompassesthe pharmaceutically acceptable salts of said glucagon agonists. In oneembodiment the polyethylene glycol chain is covalently linked toposition 21 or 24 of the glucagon peptide and has a molecular weightselected from the range of about 500 to about 40,000 Daltons. In oneembodiment the polyethylene glycol chain is covalently linked toposition 21 or 24 of the glucagon peptide and has a molecular weightselected from the range of about 500 to about 5,000 Daltons. In anotherembodiment the polyethylene glycol chain has a molecular weight of atleast about 20,000 Daltons. In one embodiment the glucagon peptidecomprises the peptide selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ IDNO: 17, SEQ ID NO: 18 and glucagon agonist derivatives thereof.

In accordance with one embodiment the glucagon peptides disclosed hereinare modified by the addition of a second peptide to the carboxy terminusof the glucagon peptide. In one embodiment a glucagon peptide iscovalently bound through a peptide bond to a second peptide, wherein thesecond peptide comprises a sequence selected from the group consistingof SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21. In one embodiment themodified glucagon peptide comprises a peptide selected from the groupconsisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ IDNO: 38, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41, wherein apolyethylene glycol chain is bound at position 21 of SEQ ID NO: 24, SEQID NO: 25, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO:40, or bound at position 24 of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO:35, SEQ ID NO: 37, SEQ ID NO: 39 and SEQ ID NO: 41, and has a molecularweight selected from the range of about 500 to about 40,000 Daltons.

In accordance with one embodiment a pharmaceutical composition isprovided comprising the novel glucagon peptides disclosed herein. In oneembodiment the pharmaceutical compositions comprise solutions that aresterilized and contained within various packages. The pharmaceuticalcompositions can be further packaged as part of a kit that includes adisposable device for administering the composition to a patient.

In accordance with one embodiment a method of rapidly treatinghypoglycemia using a pre-formulated aqueous composition is provided. Themethod comprises the step of administering an effective amount of anaqueous solution comprising a novel modified glucagon peptide of thepresent disclosure. In one embodiment the glucagon peptide is pegylatedat position 21 or 24 of the glucagon peptide and the PEG chain has amolecular weight of about 500 to about 5,000 Daltons. In one embodimentthe modified glucagon solution is prepackaged in a device that is usedto administer the composition to the patient suffering fromhypoglycemia.

In accordance with one embodiment an improved method of regulating bloodglucose levels in insulin dependent patients is provided. The methodcomprises the steps of administering insulin in an amounttherapeutically effective for the control of diabetes and administeringa novel modified glucagon peptide of the present disclosure in an amounttherapeutically effective for the prevention of hypoglycemia, whereinsaid administering steps are conducted within twelve hours of eachother. In one embodiment the glucagon peptide and the insulin areco-administered as a single composition, wherein the glucagon peptide ispegylated with a PEG chain having a molecular weight selected from therange of about 5,000 to about 40,000 Daltons

In another embodiment a method is provided for inducing the temporaryparalysis of the intestinal tract. The method comprises the step ofadministering one or more of the pegylated glucagon peptides disclosedherein to a patient.

In one embodiment a method of reducing weight gain or inducing weightloss is provided. The method comprises administering an effective amountof a composition comprising a glucagon agonist, wherein the glucagonagonist comprising a glucagon peptide selected from the group consistingof SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ IDNO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, wherein aminoacid 29 of the glucagon peptide is bound to a second peptide through apeptide bond, and said second peptide comprises the sequence of SEQ IDNO: 19, SEQ ID NO: 20 or SEQ ID NO: 21. In one embodiment the glucagonpeptide is pegylated. In one embodiment the method comprises the step ofadministering a peptide comprising the sequence of SEQ ID NO: 24, SEQ IDNO: 25, SEQ ID NO: 32 or SEQ ID NO:33, wherein a polyethylene chain iscovalently linked to amino acid position 21 of SEQ ID NO: 24 or 25, orat position 24 of SEQ ID NO: 32 or SEQ ID NO: 33.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph representing the stability of GlucagonCys²¹-maleimido PEG_(5K) at 37° C. incubated for 24, 48, 72, 96, 144 and166 hours, respectively.

FIG. 2 represents data generated from HPLC analysis of GlucagonCys²¹-maleimido PEG_(5K) at pH 5 incubated at 37° C. for 24, 72 or 144hours, respectively.

DETAILED DESCRIPTION

Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

As used herein the term “pharmaceutically acceptable salt” refers tosalts of compounds that retain the biological activity of the parentcompound, and which are not biologically or otherwise undesirable. Manyof the compounds disclosed herein are capable of forming acid and/orbase salts by virtue of the presence of amino and/or carboxyl groups orgroups similar thereto.

Pharmaceutically acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

As used herein, the term “treating” includes prophylaxis of the specificdisorder or condition, or alleviation of the symptoms associated with aspecific disorder or condition and/or preventing or eliminating saidsymptoms.

As used herein an “effective” amount or a “therapeutically effectiveamount” of a glucagon peptide refers to a nontoxic but sufficient amountof the peptide to provide the desired effect. For example one desiredeffect would be the prevention or treatment of hypoglycemia. The amountthat is “effective” will vary from subject to subject, depending on theage and general condition of the individual, mode of administration, andthe like. Thus, it is not always possible to specify an exact “effectiveamount.” However, an appropriate “effective” amount in any individualcase may be determined by one of ordinary skill in the art using routineexperimentation.

The term, “parenteral” means not through the alimentary canal but bysome other route such as subcutaneous, intramuscular, intraspinal, orintravenous.

A “glucagon peptide” as used herein includes any peptide comprising,either the amino acid sequence of SEQ ID NO: 1, or any derivative of theamino acid sequence of SEQ ID NO: 1, including amino acid substitutions,or post translational modifications (e.g. methylation, acylation,ubiquitination and the like) of the peptide, that stimulates glucagon orGLP-1 receptor activity, as measured by cAMP production using the assaydescribed in Example 13.

The term “glucagon agonist” refers to a complex comprising a glucagonpeptide.

As used herein a “glucagon agonist derivative” is a glucagon peptidethat has been modified to include one or more conservative amino acidsubstitutions at one or more of positions 2, 5, 7, 10, 11, 12, 13, 14,16, 17, 18, 19, 20, 21, 24, 27, 28 or 29.

As used herein an amino acid “substitution” refers to the replacement ofone amino acid residue by a different amino acid residue.

As used herein, the term “conservative amino acid substitution” isdefined herein as exchanges within one of the following five groups:

I. Small aliphatic, nonpolar or slightly polar residues:

-   -   Ala, Ser, Thr, Pro, Gly;

II. Polar, negatively charged residues and their amides:

-   -   Asp, Asn, Glu, Gln;

III. Polar, positively charged residues:

-   -   His, Arg, Lys; Ornithine (Orn)

IV. Large, aliphatic, nonpolar residues:

-   -   Met, Leu, Ile, Val, Cys, Norleucine (Nle), homocysteine

V. Large, aromatic residues:

-   -   Phe, Tyr, Trp, acetyl phenylalanine

As used herein the general term “polyethylene glycol” or “PEG”, refersto mixtures of condensation polymers of ethylene oxide and water, in abranched or straight chain, represented by the general formulaH(OCH₂CH₂)_(n)OH, wherein n is at least 9. Absent any furthercharacterization, the term is intended to include polymers of ethyleneglycol with an average total molecular weight selected from the range of500 to 40,000 Daltons. “polyethylene glycol” or “PEG” is used incombination with a numeric suffix to indicate the approximate averagemolecular weight thereof. For example, PEG-5,000 refers to polyethyleneglycol having a total molecular weight average of about 5,000.

As used herein the term “pegylated” and like terms refers to a compoundthat has been modified from its native state by linking a polyethyleneglycol polymer to the compound. A “pegylated glucagon peptide” is aglucagon peptide that has a PEG chain covalently bound to the glucagonpeptide.

As used herein a general reference to a peptide is intended to encompasspeptides that have modified amino and carboxy termini. For example, anamino acid chain comprising an amide group in place of the terminalcarboxylic acid is intended to be encompassed by an amino acid sequencedesignating the standard amino acids.

As used herein a “linker” is a bond, molecule or group of molecules thatbinds two separate entities to one another. Linkers may provide foroptimal spacing of the two entities or may further supply a labilelinkage that allows the two entities to be separated from each other.Labile linkages include photocleavable groups, acid-labile moieties,base-labile moieties and enzyme-cleavable groups.

As used herein a “dimer” is a complex comprising two subunits covalentlybound to one another via a linker. The term dimer, when used absent anyqualifying language, encompasses both homodimers and heterodimers. Ahomodimer comprises two identical subunits, whereas a heterodimercomprises two subunits that differ, although the two subunits aresubstantially similar to one another.

Embodiments

One embodiment of the present invention is directed to a glucagonagonist that has been modified relative to the wild type peptide ofHis-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr(SEQ ID NO: 1) to improve the peptide's solubility and stability inaqueous solutions at physiological pH, while retaining the nativepeptide's biological activity. In accordance with one embodiment,applicants have found that introduction of hydrophilic groups atpositions 16, 17, 20, 21, 24 and 29 of the native peptide can improvethe solubility and stability of the resulting glucagon analog insolutions having a physiological pH. More particularly, in oneembodiment the glucagon peptide is modified to comprise one or morehydrophilic groups covalently linked to the side chains of amino acidspresent at positions 21 and 24 of the glucagon peptide, and in oneembodiment the hydrophilic group is PEG. In one embodiment the glucagonpeptide comprises a sequence selected from the group consisting of SEQID NO: 45 and glucagon agonist derivatives of SEQ ID NO: 45, with theproviso that when the amino acid at position 21 is Asp the amino acid atposition 24 is not Gln, and when the amino acid at position 24 is Glnthe amino acid at position 21 is not Asp, wherein one or morehydrophilic groups covalently linked to the side chains of amino acidspresent at positions 21 and 24 of the glucagon peptide, and in oneembodiment the hydrophilic group is PEG.

In accordance with one embodiment, the native glucagon peptide of SEQ IDNO: 1 is modified to contain one or more amino acid substitution atpositions 16, 17, 20, 21, 24 and/or 29, wherein the native amino acid issubstituted with an amino acid having a side chain suitable forcrosslinking with hydrophilic moieties, including for example, PEG. Thenative peptide can be substituted with a naturally occurring amino acidor a synthetic (non-naturally occurring) amino acid. Synthetic ornon-naturally occurring amino acids refer to amino acids that do notnaturally occur in vivo but which, nevertheless, can be incorporatedinto the peptide structures described herein.

In one embodiment, a glucagon agonist is provided wherein the nativeglucagon peptide sequence has been modified to contain a naturallyoccurring or synthetic amino acid in at least one of positions 16, 17,20, 21, 24 and 29 of the native sequence, wherein the amino acidsubstitute further comprises a hydrophilic moiety. In one embodiment oneor more amino acids at position 16, 17, 20, 21, 24 and 29 of the nativepeptide are substituted with an amino acid selected from the groupconsisting of lysine, cysteine, ornithine, homocysteine and acetylphenylalanine, wherein the substituting amino acid further comprises ahydrophilic moiety covalently bound to the side chain of the amino acid.In one embodiment the substitution is at position 21 or 24, and in afurther embodiment the hydrophilic moiety is a PEG chain.

In one embodiment the native glucagon peptide is substituted with atleast one cysteine residue, wherein the side chain of the cysteineresidue is further modified with a thiol reactive reagent, including forexample, maleimido, vinyl sulfone, 2-pyridylthio, haloalkyl, andhaloacyl. These thiol reactive reagents may contain carboxy, keto,hydroxyl, and ether groups as well as other hydrophilic moieties such aspolyethylene glycol units. In an alternative embodiment, the nativeglucagon peptide is substituted with lysine, and the side chain of thesubstituting lysine residue is further modified using amine reactivereagents such as active esters (succinimido, anhydride, etc) ofcarboxylic acids or aldehydes of hydrophilic moieties such aspolyethylene glycol.

It has been reported that certain positions of the native glucagonpeptide can be modified while retaining at least some of the activity ofthe parent peptide. Accordingly, one or more of the amino acids locatedat positions at positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19,20, 21, 24, 27, 28 or 29 of the peptide of SEQ ID NO: 1 can besubstituted with an amino acid different from that present in the nativeglucagon peptide, and still retain the biological activity of the nativeglucagon. In accordance with one embodiment the lysine residue atposition 12 of the native peptide is substituted with arginine and asingle lysine substitution is inserted for the amino acid present atposition 16, 17, 20, 21, 24 or 29. In another embodiment the methionineresidue present at position 27 of the native peptide is changed toleucine or norleucine to prevent oxidative degradation of the peptide.

In one embodiment a glucagon peptide is provided that comprises apolyethylene glycol chain covalently bound to the side chain of an aminoacid present at position 16, 17, 20, 21, 24 or 29, wherein the glucagonpeptide further comprises one, two or three amino acid substitutions atpositions selected from positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17,18, 19, 20, 21, 24, 27, 28 or 29. In one embodiment the substitutions atpositions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19, 20, 27, 28 or 29are conservative amino acid substitutions. In one embodiment the aminoacid present at position 16, 17, 20, 21, 24 or 29 of the native peptideis substituted with cysteine or lysine. However, in one embodiment anamino acid substitution (using a natural or synthetic amino acid) ismade at position 16, 17, 20, 21, 24 or 29, wherein the substitute aminoacid allows for the covalent attachment of a PEG chain to the amino acidside chain. In one embodiment the substitution is made at position 21and/or 24.

In one embodiment an improved glucagon agonist is provided havingsuperior stability and solubility in aqueous solutions at physiologicalpH. In this embodiment the glucagon peptide is modified to comprise apolyethylene glycol chain linked to an amino acid side chain of an aminoacid located at positions 2, 5, 7, 10, 11, 12, 13, 14, 16, 17, 18, 19,20, 27, 28 or 29 of the native peptide. More particularly, in oneembodiment the polyethylene glycol chain is covalently bound to an aminoacid side chain at position 16, 17, 20, 21, 24 or 29 of the glucagonpeptide, in one embodiment the polyethylene glycol chain is bound to anamino acid side chain at position 16, 21 or 24, and in one embodimentthe polyethylene glycol chain is covalently bound to the side chain ofamino acid 21 or 24.

The polyethylene glycol chain may be in the form of a straight chain orit may be branched. In accordance with one embodiment the polyethyleneglycol chain has an average molecular weight selected from the range ofabout 500 to about 10,000 Daltons. In one embodiment the polyethyleneglycol chain has an average molecular weight selected from the range ofabout 1,000 to about 5,000 Daltons. In one embodiment the polyethyleneglycol chain has an average molecular weight selected from the range ofabout 2,000 to about 5,000 Daltons. In one embodiment the polyethyleneglycol chain has an average molecular weight selected from the range ofabout 4,000 to about 5,000 Daltons.

In accordance with one embodiment the modified glucagon peptidecomprises two or more polyethylene chains covalently bound to theglucagon peptide wherein the total molecular weight of the glucagonchains is about 1,000 to about 5,000 Daltons. In one embodiment thepegylated glucagon agonist comprises a peptide selected from the groupconsisting of SEQ ID NO: 12, SEQ ID NO: 22 and SEQ ID NO: 23 or aglucagon agonist derivative of SEQ ID NO: 12, SEQ ID NO: 22 or SEQ IDNO: 23, wherein a PEG chain is covalently linked to the amino acidresidue at position 21 and at position 24, and wherein the combinedmolecular weight of the two PEG chains is about 1,000 to about 5,000Daltons.

In accordance with one embodiment a glucagon agonist is providedcomprising a modified glucagon peptide selected from the groupconsisting of:

(SEQ ID NO: 5) NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val- Gln-Trp-Leu-Leu-Asn-Thr(SEQ ID NO: 44) NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val- Gln-Trp-Leu-Nle-Asn-Thr(SEQ ID NO: 2) NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Xaa-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Xaa-Phe-Val-Gln-Trp-Leu-Xaa-Asn-Thr-R, (SEQ ID NO: 3)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Xaa-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Xaa-Trp-Leu-Xaa-Asn-Thr-R and (SEQ ID NO: 4)NH₂-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Xaa-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Xaa-Phe-Val-Xaa-Trp-Leu-Xaa-Asn-Thr-R,wherein Xaa at position 12=Lys or Arg, Xaa at positions 21 and 24 areindependently selected from the group consisting of Lys, Cys, Orn,homocysteine and acetyl phenylalanine, Xaa at position 27=Met, Leu orNle, and R is COOH or CONH₂, wherein the peptide is pegylated atposition 21 for SEQ ID NO: 2, position 24 for SEQ ID NO: 3 and atpositions 21 and 24 of SEQ ID NO: 4. In accordance with one embodimentXaa at position 27 for SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 isLeu or Nle. In accordance with one embodiment the peptide comprises SEQID NO: 2 or SEQ ID NO: 3. In accordance with one embodiment the peptidecomprises a sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ IDNO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, wherein the peptide ispegylated at position 21 for SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15and SEQ ID NO: 17, and pegylated at position 24 for SEQ ID NO: 11, SEQID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18. In one embodiment theglucagon agonist comprises the peptide of SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8 and SEQ ID NO: 9. In one embodiment the terminal amino acidof the glucagon peptides of the present invention have an amide group inplace of the carboxylic acid group that is present on the native aminoacid.

As described in detail in the Examples, the glucagon agonists of thepresent invention have enhanced biophysical stability and aqueoussolubility while retaining the bioactivity of the native peptide, bothin terms of potency and selectivity at the glucagon and GLP-1 receptors.Accordingly, the glucagon agonists of the present invention are believedto be suitable for any use that has previously been described for thenative glucagon peptide. Accordingly, the modified glucagon peptidesdescribed herein can be used to treat hypoglycemia, to induce temporaryparalysis of the gut for radiological uses, to reduce and maintain bodyweight, or treat other metabolic diseases that result from low bloodlevels of glucagon.

One aspect of the present disclosure is directed to a pre-formulatedaqueous solution of the presently disclosed glucagon agonist for use intreating hypoglycemia. The improved stability and solubility of theagonist compositions described herein allow for the preparation ofpre-formulated aqueous solutions of glucagon for rapid administrationand treatment of hypoglycemia. In one embodiment a solution comprising apegylated glucagon agonist is provided for administration to a patientsuffering from hypoglycemia, wherein the total molecular weight of thePEG chains linked to the pegylated glucagon agonist is between about 500to about 5,000 Daltons. In one embodiment the pegylated glucagon agonistcomprises a peptide selected from the group consisting of SEQ ID NO: 2,SEQ ID NO: 3, and SEQ ID NO: 4, and glucagon agonist derivatives of SEQID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, wherein the side chain of anamino acid residue at position 21 and/or 24 of said glucagon peptide iscovalently bound to the polyethylene glycol chain. In one embodiment,the pegylated glucagon agonist comprises the peptide of SEQ ID NO: 2,wherein the amino acid residue at position 21 of the peptide iscovalently linked to polyethylene glycol. In one embodiment, thepegylated glucagon agonist comprises the peptide of SEQ ID NO: 3,wherein the amino acid residue at position 24 of the peptide iscovalently linked to polyethylene glycol. In another embodiment thepegylated glucagon agonist comprises the peptide of SEQ ID NO: 7 or SEQID NO. 8. In a further embodiment, the pegylated glucagon agonistcomprises the peptide of SEQ ID NO: 22 or SEQ ID NO: 23, wherein a PEGchain is covalently linked to the amino acid residue at position 21 andat position 24, wherein the combined molecular weight of the two PEGchains is about 1,000 to about 5,000 Daltons.

The method of treating hypoglycemia in accordance with the presentinvention comprises the steps of administering the presently disclosedglucagon agonists to a patient using any standard route ofadministration, including parenterally, such as intravenously,subcutaneously or intramuscularly, transdermally, rectally, orally,nasally or by inhalation. In one embodiment the composition isadministered subcutaneously or intramuscularly. In one embodiment, thecomposition is administered parenterally and the glucagon composition isprepackaged in a syringe. In one embodiment the glucagon composition tobe administered to treat an individual suffering from hypoglycemia isprovided as two separated solutions. The first solution comprises theglucagon agonist in an aqueous solution at a pH of about 4.5 to about5.5. In one embodiment the first solution has a pH of about 5.0. Thesecond aqueous solution is at a pH greater than 7.0 such that when thefirst solution is mixed with the second solution the pH of the resultingmixture is approximately at physiological pH. In one embodiment, aftermixture of the first and second solutions, the pH of the resultingmixture is about 7.4. In one embodiment the first and second solutionsare contained within a single vessel and separated from one another by avalve or seal wherein upon opening of the valve, or breakage of theseal, the two solutions mix to provide a composition comprising aglucagon peptide and pharmaceutically acceptable carrier wherein the pHof the composition is at a physiologically acceptable pH. In this mannerthe vessel comprising the two solutions can be stored for long periodsof time. At a time of need the two solutions can be mixed and rapidlyadministered to the patient.

Surprisingly, applicants have discovered that pegylated glucagonpeptides can be prepared that retain the parent peptide's bioactivityand specificity. However, increasing the length of the PEG chain, orattaching multiple PEG chains to the peptide, such that the totalmolecular weight of the linked PEG is greater than 5,000 Daltons, beginsto delay the time action of the modified glucagon. In accordance withone embodiment, a glucagon peptide is provided wherein the peptidecomprises one or more polyethylene glycol chains, wherein the totalmolecular weight of the linked PEG is greater than 5,000 Daltons, and inone embodiment is greater than 10,000 Daltons. Such modified glucagonpeptides have a delayed time of activity but without loss of thebioactivity. Accordingly, such compounds can be administeredprophylactically to extend the effect of the administered glucagonpeptide.

In one embodiment the pegylated glucagon agonist comprises a peptideselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3 and SEQID NO: 4, and glucagon agonist derivatives of SEQ ID NO: 2, SEQ ID NO: 3and SEQ ID NO: 4, wherein the side chain of an amino acid residue atposition 21 and/or 24 of said glucagon peptide is covalently bound toone or more polyethylene glycol chains having a combined molecularweight of greater than about 10,000 Daltons, and in one embodiment themolecular weight of the PEG chain(s) is greater than 10,000 and lessthan or equal to 40,000 Daltons. In one embodiment, the pegylatedglucagon agonist comprises the peptide of SEQ ID NO: 2, wherein an aminoacid residue at position 21 of the peptide is covalently linked to apolyethylene glycol chain having a molecular weight selected from therange of about 10,000 to about 40,000 Daltons. In one embodiment, thepegylated glucagon agonist comprises the peptide of SEQ ID NO: 3, andwherein an amino acid residue at position 24 of the peptide iscovalently linked to a polyethylene glycol chain having a molecularweight selected from the range of about 10,000 to about 40,000 Daltons.In another embodiment the pegylated glucagon agonist comprises thepeptide of SEQ ID NO: 7 or SEQ ID NO. 8, wherein the covalently linkedPEG chain has a molecular weight of at least about 10,000 Daltons, andin one embodiment the molecular weight of the PEG is selected from therange of about 20,000 to about 40,000 Daltons. In another embodiment thepegylated glucagon agonist comprises the peptide of SEQ ID NO: 22 or SEQID NO: 23, wherein a PEG chain is covalently linked to the amino acidresidue at position 21 and at position 24, wherein the combinedmolecular weight of the two PEG chains is at least about 10,000 Daltons.

Glucagon peptides that have been modified to be covalently bound to aPEG chain having a molecular weight of greater than 10,000 Daltons canbe administered in conjunction with insulin to buffer the actions ofinsulin and help to maintain stable blood glucose levels in diabetics.The modified glucagon peptides of the present disclosure can beco-administered with insulin as a single composition, simultaneouslyadministered as separate solutions, or alternatively, the insulin andthe modified glucagon peptide can be administered at different timerelative to one another. In one embodiment the composition comprisinginsulin and the composition comprising the modified glucagon peptide areadministered within 12 hours of one another. The exact ratio of themodified glucagon peptide relative to the administered insulin will bedependent in part on determining the glucagon levels of the patient, andcan be determined through routine experimentation.

In accordance with one embodiment an aqueous solution is providedcomprising insulin and a modified glucagon peptide, wherein the glucagonpeptide comprises a polyethylene glycol chain covalently bound to anamino acid side chain at position 16, 17, 20, 21, 24 or 29. In oneembodiment the molecular weight of the PEG chain of the modifiedglucagon peptide is greater than 10,000 Daltons. In one embodiment thepegylated glucagon peptide comprises a peptide selected from the groupconsisting of SEQ ID NO: 2 and SEQ ID NO: 3 wherein the side chain of anamino acid residue at position 21 or 24 of said glucagon peptide iscovalently bound to the polyethylene glycol chain. In one embodiment,the pegylated glucagon agonist comprises the peptide of SEQ ID NO: 2,wherein an amino acid residue at position 21 of the peptide iscovalently linked to a polyethylene glycol chain having a molecularweight of about 10,000 to about 40,000. In one embodiment, the pegylatedglucagon agonist comprises the peptide of SEQ ID NO: 3, wherein an aminoacid residue at position 24 of the peptide is covalently linked to apolyethylene glycol chain having a molecular weight of about 10,000 toabout 40,000. In another embodiment the pegylated glucagon agonistcomprises the peptide of SEQ ID NO: 7 or SEQ ID NO. 8.

The present disclosure also encompasses glucagon fusion peptides whereina second peptide has been fused to the c-terminus of the glucagonpeptide. More particularly, the fusion glucagon peptide may comprise aglucagon agonist derivative of SEQ ID NO: 1 further comprising an aminoacid sequence of SEQ ID NO: 19 (GPSSGAPPPS), SEQ ID NO: 20 (KRNRNNIA) orSEQ ID NO: 21 (KRNR) linked to amino acid 29 of the glucagon peptide. Inone embodiment the amino acid sequence of SEQ ID NO: 19 (GPSSGAPPPS),SEQ ID NO: 20 (KRNRNNIA) or SEQ ID NO: 21 (KRNR) is bound to amino acid29 of the glucagon peptide through a peptide bond. In one embodiment theglucagon peptide portion of the glucagon fusion peptide is selected fromthe group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, SEQ IDNO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 wherein thePEG chain, when present, is selected from the range of 500 to 40,000Daltons. More particularly, in one embodiment the glucagon peptidesegment is selected from the group consisting of SEQ ID NO: 7 and SEQ IDNO: 8 wherein the PEG chain is selected from the range of 500 to 5,000.In one embodiment the glucagon fusion peptide comprises the sequence ofSEQ ID NO: 22 or SEQ ID NO: 23. In one embodiment the glucagon fusionpeptide comprises the sequence of SEQ ID NO: 24, SEQ ID NO: 25, SEQ IDNO: 32 or SEQ ID NO: 33, wherein a polyethylene chain of about 500 to5,000 Daltons is covalently linked to amino acid position 21 of SEQ IDNO: 24 or 25, or at position 24 of SEQ ID NO: 32 or SEQ ID NO: 33.

In one embodiment a glucagon fusion peptide is provided comprising asequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17 and SEQ ID NO: 18, covalently linked to the sequence of SEQ ID NO: 19(GPSSGAPPPS) or SEQ ID NO: 21, wherein the PEG chain, when present, isselected from the range of 500 to 40,000 Daltons. In one embodiment thefusion peptide comprises a glucagon peptide selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 covalently linked tothe sequence of SEQ ID NO: 19 (GPSSGAPPPS) or SEQ ID NO: 21. In anotherembodiment the fusion peptide comprises a glucagon peptide selected fromthe group consisting of SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 17 and SEQ ID NO: 18 covalently linked to thesequence of SEQ ID NO: 19 (GPSSGAPPPS) or SEQ ID NO: 21.

In one embodiment the composition comprises a sequence selected from thegroup consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ IDNO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 covalently linkedto the sequence of SEQ ID NO: 20 (KRNRNNIA). In one embodiment thefusion peptide comprises a glucagon peptide selected from the groupconsisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:16, SEQ ID NO: 17 and SEQ ID NO: 18 covalently linked to the sequence ofSEQ ID NO: 20 (KRNRNNIA). In another embodiment the fusion peptidecomprises a glucagon peptide selected from the group consisting of SEQID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 17 andSEQ ID NO: 18 covalently linked to the sequence of SEQ ID NO: 20(KRNRNNIA).

In accordance with one embodiment the modified glucagon peptidesdisclosed herein are used to induce temporary paralysis of theintestinal tract. This method has utility for radiological purposes andcomprises the step of administering an effective amount of apharmaceutical composition comprising a pegylated glucagon peptide, aglucagon peptide comprising a c-terminal extension or a dimer of suchpeptides. In one embodiment the glucagon peptide comprises a sequenceselected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ IDNO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 wherein a PEG chain, of about1,000 to 40,000 Daltons is covalently bound to an amino acid residue atposition 21 or 24. In one embodiment the glucagon peptide is selectedfrom the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 17 and SEQ ID NO: 18. In one embodiment thePEG chain has a molecular weight of about 500 to about 5,000 Daltons.

In a further embodiment the composition used to induce temporaryparalysis of the intestinal tract comprises a first modified glucagonpeptide and a second modified glucagon peptide, wherein the firstmodified peptide comprises a covalently linked PEG chain of about 500 toabout 5,000 Daltons and the second peptide comprises a covalently linkedPEG chain of about 10,000 to about 40,000 Daltons. In this embodimentthe PEG chain of each peptide is covalently bound to an amino acidresidue at either position 21 or 24 of the respective peptides, andindependent of one another.

Oxyntomodulin, a naturally occurring digestive hormone found in thesmall intestine, has been reported to cause weight loss whenadministered to rats or humans (see Diabetes 2005; 54:2390-2395).Oxyntomodulin is a 37 amino acid peptide that contains the 29 amino acidsequence of glucagon (i.e. SEQ ID NO: 1) followed by an 8 amino acidcarboxy terminal extension of SEQ ID NO: 20 (KRNRNNIA). Accordingly,applicants believe that the bioactivity of oxyntomodulin can be retained(i.e. appetite suppression and induced weight loss/weight maintenance),while improving the solubility and stability of the compound andimproving the pharmacokinetics, by substituting the glucagon peptideportion of oxyntomodulin with the modified glucagon peptides disclosedherein. In addition applicants also believe that a truncatedOxyntomodulin molecule, having the terminal four amino acids removedwill also be effective in suppressing appetite and inducing weightloss/weight maintenance.

Accordingly, the present invention also encompasses the modifiedglucagon peptides of the present invention that have a carboxy terminalextension of SEQ ID NO: 20 (KRNRNNIA) or SEQ ID NO: 21. In accordancewith one embodiment a glucagon agonist derivative of SEQ ID NO: 1further comprising the amino acid sequence of SEQ ID NO: 20 (KRNRNNIA)or SEQ ID NO: 21 is linked to amino acid 29 of the glucagon peptide isadministered to individuals to induce weight loss or prevent weightgain. In another embodiment a method of reducing weight gain or inducingweight loss in an individual comprises administering an effective amountof a composition comprising a glucagon agonist comprising a glucagonpeptide selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3or SEQ ID NO: 4, wherein amino acid 29 of the glucagon peptide is boundto a second peptide through a peptide bond, and said second peptidecomprises the sequence of SEQ ID NO: 20 (KRNRNNIA) or SEQ ID NO: 21. Inone embodiment the glucagon peptide segment of the glucagon agonist isselected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17and SEQ ID NO: 18, wherein a PEG chain of about 1,000 to 40,000 Daltonsis covalently bound to an amino acid residue at position 21 or 24. Inone embodiment the glucagon peptide segment is selected from the groupconsisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 17 and SEQ ID NO: 18 wherein the molecular weight of the PEGchain is selected from the range of 1,000 to 40,000 Daltons. Moreparticularly, in one embodiment the glucagon peptide segment of theglucagon fusion peptide is selected from the group consisting of SEQ IDNO: 7 and SEQ ID NO: 8 wherein the molecular weight of the PEG chain isselected from the range of 1,000 to 40,000. In another embodiment acomposition is administered to a patient to suppress appetite, preventweight gain and/or induce weight loss by the administration of apharmaceutical composition comprising a glucagon peptide selected fromthe group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 32, SEQID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35. In one embodiment theglucagon peptide selected from the group consisting of SEQ ID NO: 24,SEQ ID NO: 25, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ IDNO: 35 is further modified to comprise a PEG chain covalently bound toamino acid position 21 or 24. In one embodiment the molecular weight ofthe PEG chain is selected from the range of 500 to 5,000 Daltons, and inanother embodiment the glucagon peptide is selected from the groupconsisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO: 34 and SEQ ID NO: 35 wherein the molecular weight of thePEG chain is selected from the range of 10,000 to 40,000 Daltons.

Exendin-4, is a peptide made up of 39 amino acids. It is a powerfulstimulator of a receptor known as GLP-1. This peptide has also beenreported to suppress appetite and induce weight loss. Applicants havefound that the terminal sequence of Exendin-4 when added at the carboxyterminus of glucagon improves the solubility and stability of glucagonwithout compromising the bioactivity of glucagon. In one embodiment theterminal ten amino acids of Exendin-4 (i.e. the sequence of SEQ ID NO:19 (GPSSGAPPPS)) are linked to the carboxy terminus of a glucagonpeptide of the present disclosure. These fusion proteins are anticipatedto have pharmacological activity for suppressing appetite and inducingweight loss/weight maintenance. In one embodiment the terminal aminoacid of the SEQ ID NO: 19 extension comprises an amide group in place ofthe carboxy group.

In one embodiment a method of reducing weight gain or inducing weightloss in an individual comprises administering an effective amount of acomposition comprising a glucagon agonist comprising a glucagon peptideselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 andSEQ ID NO: 18 wherein amino acid 29 of the glucagon peptide is bound toa second peptide through a peptide bond, and said second peptidecomprises the sequence of SEQ ID NO: 19 (GPSSGAPPPS). In one embodimentthe glucagon peptide of the glucagon agonist is selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:17 and SEQ ID NO: 18, wherein the molecular weight of the PEG chain,when present is selected from the range of 500 to 40,000 Daltons. Inanother embodiment the glucagon peptide portion of the fusion peptidecomprises SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14,SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, wherein aPEG chain of about 1,000 to 40,000 Daltons is covalently bound to anamino acid residue at position 21 or 24. In one embodiment the glucagonpeptide segment is selected from the group consisting of SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 17 and SEQ ID NO:18, wherein the molecular weight of the PEG chain, when present isselected from the range of 500 to 40,000 Daltons. More particularly, inone embodiment the glucagon peptide is selected from the groupconsisting of SEQ ID NO: 7 and SEQ ID NO: 8 wherein the molecular weightof the PEG chain is selected from the range of 1,000 to 5,000.

In another embodiment a composition is administered to a patient tosuppress appetite, prevent weight gain and/or induce weight loss by theadministration of a pharmaceutical composition comprising a firstpegylated glucagon peptide and a second pegylated glucagon peptide,wherein the first and second peptide are fusion peptides comprising ac-terminal peptide extension comprising SEQ ID NO: 19 (GPSSGAPPPS). Thefirst pegylated glycogen peptide comprising a covalently linked PEG ofabout 500 to about 10,000 Daltons and the second pegylated glucagonpeptide comprising a covalently linked PEG chain of about 10,000 toabout 40,000 Daltons.

In accordance with one embodiment, a glucagon analogue is providedwherein a plasma protein has been covalently linked to an amino acidside chain of the glucagon peptide to improve the solubility, stabilityand/or pharmacokinetics of the glucagon peptide. For example, serumalbumin can be covalently bound to glucagon or a glucagon analogue ofthe present invention. In one embodiment the plasmid protein iscovalently bound to position 16, 17, 20 21, 24 or 29, and moreparticularly, in one embodiment the plasmid protein is bound at position21 or 24 of the glucagon peptide. In one embodiment the glucagon peptideis selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 11, SEQID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ IDNO: 39, SEQ ID NO: 40 and SEQ ID NO: 41. In one embodiment the glucagonpeptide is selected from the group consisting of SEQ ID NO: 1, SEQ IDNO: 5, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 37,SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41. In oneembodiment the glucagon peptide is selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,SEQ ID NO: 18, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO:40 and SEQ ID NO: 41. In one embodiment the glucagon analog comprises aglucagon peptide selected from the group consisting of SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, wherein amino acid 29 of theglucagon peptide is bound to a second peptide through a peptide bond,said second peptide comprising the sequence of SEQ ID NO: 19, SEQ ID NO:20 or SEQ ID NO: 21, and a plasma protein is bound to the side chain ofthe amino acid located at position 21 or 24.

The present disclosure also encompasses multimers of the modifiedglucagon peptides disclosed herein. Two or more of the modified glucagonpeptides can be linked together using standard linking agents andprocedures known to those skilled in the art. For example, dimers can beformed between two modified glucagon peptides through the use ofbifunctional thiol crosslinkers and bi-functional amine crosslinkers,particularly for the glucagon peptides that have been substituted withcysteine, lysine ornithine, homocysteine or acetyl phenylalanineresidues (e.g. SEQ ID NO: 2 and SEQ ID NO: 3). The dimer can be ahomodimer or alternatively can be a heterodimer. In one embodiment thedimer comprises a homodimer of a glucagon fusion peptide wherein theglucagon peptide portion comprises an agonist derivative of SEQ ID NO: 1and the second peptide comprising an amino acid sequence of SEQ ID NO:19 (GPSSGAPPPS), SEQ ID NO: 20 (KRNRNNIA) or SEQ ID NO: 21 (KRNR) linkedto amino acid 29 of the glucagon peptide. In another embodiment thedimer comprises a homodimer of a glucagon agonist derivative of SEQ IDNO: 1, wherein the glucagon peptide further comprises a polyethyleneglycol chain covalently bound to position 21 or 24 of the glucagonpeptide.

In accordance with one embodiment a dimer is provided comprising a firstglucagon peptide bound to a second glucagon peptide via a linker,wherein said first glucagon peptide is selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9and the second glucagon peptide is independently selected from the groupconsisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 andSEQ ID NO: 9, and pharmaceutically acceptable salts of said glucagonpolypeptides. In one embodiment the first glucagon peptide is selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 17 and SEQ ID NO: 18 and thesecond glucagon peptide is independently selected from the groupconsisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 17 and SEQ ID NO: 18. In one embodiment the first glucagonpeptide is selected from the group consisting of SEQ ID NO: 7 and SEQ IDNO: 8 and the second glucagon peptide is independently selected from thegroup consisting of SEQ ID NO: 7 and SEQ ID NO: 8.

The modified glucagon peptides of the present invention can be providedin accordance with one embodiment as part of a kit. In one embodiment akit for administering a glucagon agonist to a patient in need thereof isprovided wherein the kit comprises a modified glucagon peptide selectedfrom the group consisting of 1) a pegylated glucagon peptide, whereinthe PEG chain is covalently bound to position 16, 17, 20, 21, 24 or 29of the glucagon peptide, and the PEG chain has a molecular weight ofabout 500 to about 40,000 Daltons; 2) a glucagon fusion peptidecomprising a glucagon agonist derivative of SEQ ID NO: 1, and an aminoacid sequence of SEQ ID NO: 19 (GPSSGAPPPS), SEQ ID NO: 20 (KRNRNNIA) orSEQ ID NO: 21 (KRNR) linked to amino acid 29 of the glucagon peptide;and 3) a pegylated glucagon peptide, further comprising an amino acidsequence of SEQ ID NO: 19 (GPSSGAPPPS), SEQ ID NO: 20 (KRNRNNIA) or SEQID NO: 21 (KRNR) linked to amino acid 29 of the glucagon peptide,wherein the PEG chain covalently bound to position 16, 17, 20, 21, 24 or29 has a molecular weight of about 500 to about 40,000 Daltons. In oneembodiment the kit is provided with a device for administering theglucagon composition to a patient. The kit may further include a varietyof containers, e.g., vials, tubes, bottles, and the like. Preferably,the kits will also include instructions for use. In accordance with oneembodiment the device of the kit is an aerosol dispensing device,wherein the composition is prepackaged within the aerosol device. Inanother embodiment the kit comprises a syringe and a needle, and in oneembodiment the glucagon composition is prepackaged within the syringe.

The compounds of this invention may be prepared by standard syntheticmethods, recombinant DNA techniques, or any other methods of preparingpeptides and fusion proteins. Although certain non-natural amino acidscannot be expressed by standard recombinant DNA techniques, techniquesfor their preparation are known in the art. Compounds of this inventionthat encompass non-peptide portions may be synthesized by standardorganic chemistry reactions, in addition to standard peptide chemistryreactions when applicable.

EXAMPLES

General Synthesis Protocol:

Glucagon analogs were synthesized using HBTU-activated “Fast Boc” singlecoupling starting from 0.2 mmole of Boc Thr(OBzl)Pam resin on a modifiedApplied Biosystem 430 A peptide synthesizer. Boc amino acids and HBTUwere obtained from Midwest Biotech (Fishers, Ind.). Side chainprotecting groups used were: Arg(Tos), Asn(Xan), Asp(OcHex),Cys(pMeBzl), His(Bom), Lys(2Cl—Z), Ser(OBzl), Thr(OBzl), Tyr(2Br—Z), andTrp(CHO). The side-chain protecting group on the N-terminal His was Boc.

Each completed peptidyl resin was treated with a solution of 20%piperidine in dimethylformamide to remove the formyl group from thetryptophan. Liquid hydrogen fluoride cleavages were performed in thepresence of p-cresol and dimethyl sulfide. The cleavage was run for 1hour in an ice bath using an HF apparatus (Penninsula Labs). Afterevaporation of the HF, the residue was suspended in diethyl ether andthe solid materials were filtered. Each peptide was extracted into 30-70ml aqueous acetic acid and a diluted aliquot was analyzed by HPLC[Beckman System Gold, 0.46×5 cm Zorbax C8, 1 ml/min, 45 C, 214 nm, Abuffer=0.1% TFA, B=0.1% TFA/90% acetonitrile, gradient of 10% to 80% Bover 10 min].

Purification was done on a FPLC over a 2.2×25 cm Kromasil C18 columnwhile monitoring the UV at 214 nm and collecting 5 minute fractions. Thehomogeneous fractions were combined and lyophilized to give a productpurity of >95%. The correct molecular mass and purity were confirmedusing MALDI-mass spectral analysis.

General Pegylation Protocol: (Cys-Maleimido)

Typically, the glucagon Cys analog is dissolved in phosphate bufferedsaline (5-10 mg/ml) and 0.01M ethylenediamine tetraacetic acid is added(10-15% of total volume). Excess (2-fold) maleimido methoxyPEG reagent(Nektar) is added and the reaction stirred at room temp while monitoringreaction progress by HPLC. After 8-24 hrs, the reaction mixture, isacidified and loaded onto a preparative reverse phase column forpurification using 0.1% TFA/acetonitrile gradient. The appropriatefractions were combined and lyophilized to give the desired pegylatedderivatives.

Example 1

Synthesis of Glucagon Cys¹⁷(1-29) and Similar MonoCys Analogs

0.2 mmole Boc Thr(OBzl) Pam resin (SynChem Inc) in a 60 ml reactionvessel and the following sequence was entered and run on a modifiedApplied Biosystems 430A Peptide Synthesizer using FastBoc HBTU-activatedsingle couplings.

HSQGTFTSDYSKYLDSCRAQDFVQWLMNT (SEQ ID NO: 28)The following side chain protecting groups were used: Arg(Tos),Asp(OcHex), Asn(Xan), Cys(pMeBzl), Glu(OcHex), His(Boc), Lys(2Cl—Z),Ser(Bzl), Thr(Bzl), Trp(CHO), and Tyr(Br—Z). The completed peptidylresin was treated with 20% piperidine/dimethylformamide to remove theTrp formyl protection then transferred to an HF reaction vessel anddried in vacuo. 1.0 ml p-cresol and 0.5 ml dimethyl sulfide were addedalong with a magnetic stir bar. The vessel was attached to the HFapparatus (Pennisula Labs), cooled in a dry ice/methanol bath,evacuated, and aprox. 10 ml liquid hydrogen fluoride was condensed in.The reaction was stirred in an ice bath for 1 hr then the HF was removedin vacuo. The residue was suspended in ethyl ether; the solids werefiltered, washed with ether, and the peptide extracted into 50 mlaqueous acetic acid. An analytical HPLC was run [0.46×5 cm Zorbax C8, 1ml/min, 45 C, 214 nm, A buffer of 0.1% TFA, B buffer of 0.1% TFA/90%ACN, gradient=10% B to 80% B over 10 min.] with a small sample of thecleavage extract. The remaining extract was loaded onto a 2.2×25 cmKromasil C18 preparative reverse phase column and an acetonitrilegradient was run using a Pharmacia FPLC system. 5 min fractions werecollected while monitoring the UV at 214 nm (2.0 A). A=0.1% TFA, B=0.1%TFA/50% acetonitrile. Gradient=30% B to 100% B over 450 min.

The fractions containing the purest product (48-52) were combinedfrozen, and lyophilized to give 30.1 mg. An HPLC analysis of the productdemonstrated a purity of >90% and MALDI mass spectral analysisdemonstrated the desired mass of 3429.7. Glucagon Cys²¹, Glucagon Cys²⁴,and Glucagon Cys²⁹ were similarly prepared.

Example 2

Synthesis of Glucagon-Cex and Other C-Terminal Extended Analogs.

285 mg (0.2 mmole) methoxybenzhydrylamine resin (Midwest Biotech) wasplaced in a 60 ml reaction vessel and the following sequence was enteredand run on a modified Applied Biosystems 430A peptide synthesizer usingFastBoc HBTU-activated single couplings.

(SEQ ID NO: 29) HSQGTFTSDYSKYDSRRAQDFVQWLMNTGPSSGAPPPSThe following side chain protecting groups were used: Arg(Tos),Asp(OcHex), Asn(Xan), Cys(pMeBzl), Glu(OcHex), His(Boc), Lys(2Cl—Z),Ser(Bzl), Thr(Bzl), Trp(CHO), and Tyr(Br13 Z). The completed peptidylresin was treated with 20% piperidine/dimethylformamide to remove theTrp formyl protection then transferred to HF reaction vessel and driedin vacuo. 1.0 ml p-cresol and 0.5 ml dimethyl sulfide were added alongwith a magnetic stir bar. The vessel was attached to the HF apparatus(Pennisula Labs), cooled in a dry ice/methanol bath, evacuated, andaprox. 10 ml liquid hydrogen fluoride was condensed in. The reaction wasstirred in an ice bath for 1 hr then the HF was removed in vacuo. Theresidue was suspended in ethyl ether; the solids were filtered, washedwith ether, and the peptide extracted into 50 ml aqueous acetic acid. Ananalytical HPLC was run [0.46×5 cm Zorbax C8, 1 ml/min, 45 C, 214 nm, Abuffer of 0.1% TFA, B buffer of 0.1% TFA/90% ACN, gradient=10% B to 80%B over 10 min.] on an aliquot of the cleavage extract. The extract wasloaded onto a 2.2×25 cm Kromasil C18 preparative reverse phase columnand an acetonitrile gradient was run for elution using a Pharmacia FPLCsystem. 5 min fractions were collected while monitoring the UV at 214 nm(2.0 A). A=0.1% TFA, B=0.1% TFA/50% acetonitrile. Gradient=30% B to 100%B over 450 min. Fractions 58-65 were combined, frozen and lyophilized togive 198.1 mg.

HPLC analysis of the product showed a purity of greater than 95%. MALDImass spectral analysis showed the presence of the desired theoreticalmass of 4316.7 with the product as a C-terminal amide. Oxyntomodulin andoxyntomodulin-KRNR were similarly prepared as the C-terminal carboxylicacids starting with the appropriately loaded PAM-resin.

Example 3

Glucagon Cys¹⁷ Mal-PEG-5K

15.1 mg of Glucagon Cys¹⁷(1-29) and 27.3 mg methoxy poly(ethyleneglycol)maleimide avg. M.W. 5000 (mPEG-Mal-5000, Nektar Therapeutics) weredissolved in 3.5 ml phosphate buffered saline (PBS) and 0.5 ml 0.01Methylenediamine tetraacetic acid (EDTA) was added. The reaction wasstirred at room temperature and the progress of the reaction wasmonitored by HPLC analysis [0.46×5 cm Zorbax C8, 1 ml/min, 45 C, 214 nm(0.5 A), A=0.1% TFA, B=0.1% TFA/90% ACN, gradient=10% B to 80% B over 10min.].

After 5 hours, the reaction mixture was loaded onto 2.2×25 cm KromasilC18 preparative reverse phase column. An acetonitrile gradient was runon a Pharmacia FPLC while monitoring the UV wavelength at 214 nm andcollecting 5 min fractions. A=0.1% TFA, B=0.1% TFA/50% acetonitrile,gradient=30% B to 100% B over 450 min. The fractions corresponding tothe product were combined, frozen and lyophilized to give 25.9 mg.

This product was analyzed on HPLC [0.46×5 cm Zorbax C8, 1 ml/min, 45 C,214 nm (0.5 A), A=0.1% TFA, B=0.1% TFA/90% ACN, gradient=10% B to 80% Bover 10 min.] which showed a purity of aprox. 90%. MALDI (matrixassisted laser desorption ionization) mass spectral analysis showed abroad mass range (typical of PEG derivatives) of 8700 to 9500. Thisshows an addition to the mass of the starting glucagon peptide (3429) ofapproximately 5,000 a.m.u.

Example 4

Glucagon Cys²¹ Mal-PEG-5K

21.6 mg of Glucagon Cys²¹(1-29) and 24 mg mPEG-MAL-5000 (NektarTherapeutics) were dissolved in 3.5 ml phosphate buffered saline (PBS)and 0.5 ml 0.01M ethylene diamine tetraacetic acid (EDTA) was added. Thereaction was stirred at room temp. After 2 hrs, another 12.7 mg ofmPEG-MAL-5000 was added. After 8 hrs, the reaction mixture was loadedonto a 2.2×25 cm Vydac C18 preparative reverse phase column and anacetonitrile gradient was run on a Pharmacia FPLC at 4 ml/min whilecollecting 5 min fractions. A=0.1% TFA, B=0.1% TFA/50% ACN. Gradient=20%to 80% B over 450 min.

The fractions corresponding to the appearance of product were combinedfrozen and lyophilized to give 34 mg. Analysis of the product byanalytical HPLC [0.46×5 cm Zorbax C8, 1 ml/min, 45 C, 214 nm (0.5 A),A=0.1% TFA, B=0.1% TFA/90% ACN, gradient=10% B to 80% B over 10 min.]showed a homogeneous product that was different than starting glucagonpeptide. MALDI (matrix assisted laser desorption ionization) massspectral analysis showed a broad mass range (typical of PEG derivatives)of 8700 to 9700. This shows an addition to the mass of the startingglucagon peptide (3470) of approximately 5,000 a.m.u.

Example 5

Glucagon Cys²⁴ Mal-PEG-5K

20.1 mg Glucagon C²⁴(1-29) and 39.5 mg mPEG-Mal-5000 (NektarTherapeutics) were dissolved in 3.5 ml PBS with stirring and 0.5 ml0.01M EDTA was added. The reaction was stirred at room temp for 7 hrs,then another 40 mg of mPEG-Mal-5000 was added. After approximately 15hr, the reaction mixture was loaded onto a 2.2×25 cm Vydac C18preparative reverse phase column and an acetonitrile gradient was runusing a Pharmacia FPLC. 5 min. fractions were collected while monitoringthe UV at 214 nm (2.0 A). A buffer=0.1% TFA, B buffer=0.1% TFA/50% ACN,gradient=30% B to 100% B over 450 min. The fractions corresponding toproduct were combined, frozen and lyophilized to give 45.8 mg. MALDImass spectral analysis showed a typical PEG broad signal with a maximumat 9175.2 which is approximately 5,000 a.m.u. more than Glucagon C²⁴(3457.8).

Example 6

Glucagon Cys²⁴ Mal-PEG-20K

25.7 mg of Glucagon Cys²⁴(1-29) and 40.7 mg mPEG-Mal-20K (NektarTherapeutics) were dissolved in 3.5 ml PBS with stirring at room temp.and 0.5 ml 0.01M EDTA was added. After 6 hrs, the ratio of startingmaterial to product was aprox. 60:40 as determined by HPLC. Another 25.1mg of mPEG-Mal-20K was added and the reaction allowed to stir another 16hrs. The product ratio had not significantly improved, so the reactionmixture was loaded onto a 2.2×25 cm Kromasil C18 preparative reversephase column and purified on a Pharmacia FPLC using a gradient of 30% Bto 100% B over 450 min. A buffer=0.1% TFA, B buffer=0.1% TFA/50% ACN,flow=4 ml/min, and 5 min fractions were collected while monitoring theUV at 214 nm (2.0 A). The fractions containing homogeneous product werecombined, frozen and lyophilized to give 25.7 mg. Purity as determinedby analytical HPLC was ˜90%. A MALDI mass spectral analysis showed abroad peak from 23,000 to 27,000 which is approximately 20,000 a.m.u.more than starting Glucagon C²⁴ (3457.8).

Example 7

Glucagon Cys²⁹ Mal-PEG-5K

20.0 mg of Glucagon Cys²⁹(1-29) and 24.7 mg mPEG-Mal-5000 (NektarTherapeutics) were dissolved in 3.5 ml PBS with stirring at roomtemperature and 0.5 ml 0.01M EDTA was added. After 4 hr, another 15.6 mgof mPEG-Mal-5000 was added to drive the reaction to completion. After 8hrs, the reaction mixture was loaded onto a 2.2×25 cm Vydac C18preparative reverse phase column and an acetonitrile gradient was run ona Pharmacia FPLC system. 5 min fractions were collected while monitoringthe UV at 214 nm (2.0 A). A=0.1% TFA, B=0.1% TFA/50% ACN. Fractions75-97 were combined frozen and lyophilized to give 40.0 mg of productthat is different than recovered starting material on HPLC (fractions58-63). Analysis of the product by analytical HPLC [0.46×5 cm Zorbax C8,1 ml/min, 45 C, 214 nm (0.5 A), A=0.1% TFA, B=0.1% TFA/90% ACN,gradient=10% B to 80% B over 10 min.] showed a purity greater than 95%.MALDI mass spectral analysis showed the presence of a PEG component witha mass range of 8,000 to 10,000 (maximum at 9025.3) which is 5,540a.m.u. greater than starting material (3484.8).

Example 8

Glucagon Cys²⁴ (2-butyrolactone)

To 24.7 mg of Glucagon Cys²⁴(1-29) was added 4 ml 0.05M ammoniumbicarbonate/50% acetonitrile and 5.5 ul of a solution of2-bromo-4-hydroxybutyric acid-γ-lactone (100 ul in 900 ul acetonitrile).After 3 hrs of stirring at room temperature, another 105 ul of lactonesolution was added to the reaction mixture which was stirred another 15hrs. The reaction mixture was diluted to 10 ml with 10% aqueous aceticacid and was loaded onto a 2.2×25 cm Kromasil C18 preparative reversephase column. An acetonitrile gradient (20% B to 80% B over 450 min) wasrun on a Pharmacia FPLC while collecting 5 min fractions and monitoringthe UV at 214 nm (2.0 A). Flow=4 ml/min, A=0.1% TFA, B=0.1% TFA/50% ACN.Fractions 74-77 were combined frozen and lyophilized to give 7.5 mg.HPLC analysis showed a purity of 95% and MALDI mass spect analysisshowed a mass of 3540.7 or 84 mass units more than starting material.This result consistent with the addition of a single butyrolactonemoiety.

Example 9

Glucagon Cys²⁴(S-carboxymethyl)

18.1 mg of Glucagon Cys²⁴(1-29) was dissolved in 9.4 ml 0.1M sodiumphosphate buffer (pH=9.2) and 0.6 ml bromoacetic acid solution (1.3mg/ml in acetonitrile) was added. The reaction was stirred at roomtemperature and the reaction progress was followed by analytical HPLC.After 1 hr another 0.1 ml bromoacetic acid solution was added. Thereaction was stirred another 60 min. then acidified with aqueous aceticacid and was loaded onto a 2.2×25 cm Kromasil C18 preparative reversephase column for purification. An acetonitrile gradient was run on aPharmacia FPLC (flow=4 ml/min) while collecting 5 min fractions andmonitoring the UV at 214 nm (2.0 A). A=0.1% TFA, B=0.1% TFA/50% ACN.Fractions 26-29 were combined frozen and lyophilized to give

several mg of product. Analytical HPLC showed a purity of 90% and MALDImass spectral analysis confirmed a mass of 3515 for the desired product.

Example 10

Glucagon Cys²⁴ Maleimido, PEG-3.4K-dimer

16 mg Glucagon Cys²⁴ and 1.02 mg Mal-PEG-Mal-3400,poly(ethyleneglycol)-bis-maleimide avg. M.W. 3400, (Nektar Therapeutics)were dissolved in 3.5 phosphate buffered saline and 0.5 ml 0.01M EDTAand the reaction was stirred at room temperature. After 16 hrs, another16 mg of Glucagon Cys²⁴ was added and the stirring continued. Afterapproximately 40 hrs, the reaction mixture was loaded onto a PharmaciaPepRPC 16/10 column and an acetonitrile gradient was run on a PharmaciaFPLC while collecting 2 min fractions and monitoring the UV at 214 nm(2.0 A). Flow=2 ml/min, A=0.1% TFA, B=0.1% TFA/50% ACN. Fractions 69-74were combined frozen and lyophilized to give 10.4 mg. Analytical HPLCshowed a purity of 90% and MALDI mass spectral analysis shows acomponent in the 9500-11,000 range which is consistent with the desireddimer.

Example 11

Glucagon Solubility Assays:

A solution (1 mg/ml or 3 mg/ml) of glucagon (or an analog) is preparedin 0.01N HCl. 100 ul of stock solution is diluted to 1 ml with 0.01N HCland the UV absorbance (276 nm) is determined. The pH of the remainingstock solution is adjusted to pH7 using 200-250 ul 0.1M Na₂HPO₄ (pH9.2).The solution is allowed to stand overnight at 4° C. then centrifuged.100 ul of supernatant is then diluted to 1 ml with 0.01N HCl, and the UVabsorbance is determined (in duplicate).

The initial absorbance reading is compensated for the increase in volumeand the following calculation is used to establish percent solubility:

${\frac{{Final}\mspace{14mu}{Absorbance}}{{Initial}\mspace{14mu}{Absorbance}} \times 100} = {{percent}\mspace{14mu}{soluble}}$Results are shown in Table 1 wherein Glucagon-Cex represents wild typeglucagon (SEQ ID NO: 1) plus a carboxy terminal addition of SEQ ID NO:19 and Glucagon-Cex R¹² represents SEQ ID NO: 43 plus a carboxy terminaladdition of SEQ ID NO: 19.

TABLE 1 Solubility date for glucagon analogs Analog Percent SolubleGlucagon 16 Glucagon-Cex, R12 104 Glucagon-Cex 87 Oxyntomodulin 104Glucagon, Cys17PEG5K 94 Glucagon, Cys21PEG5K 105 Glucagon, Cys24PEG5K133

Example 12

Glucagon Receptor Binding Assay

The affinity of peptides to the glucagon receptor was measured in acompetition binding assay utilizing scintillation proximity assaytechnology. Serial 3-fold dilutions of the peptides made inscintillation proximity assay buffer (0.05 M Tris-HCl, pH 7.5, 0.15 MNaCl, 0.1% w/v bovine serum albumin) were mixed in 96 well white/clearbottom plate (Corning Inc., Acton, Mass.) with 0.05 nM(3-[¹²⁵I]-iodotyrosyl) Tyr10 glucagon (Amersham Biosciences, Piscataway,N.J.), 1-6 micrograms per well, plasma membrane fragments prepared fromcells over-expressing human glucagon receptor, and 1 mg/wellpolyethyleneimine-treated wheat germ agglutinin type A scintillationproximity assay beads (Amersham Biosciences, Piscataway, N.J.). Upon 5min shaking at 800 rpm on a rotary shaker, the plate was incubated 12 hat room temperature and then read on MicroBeta 1450 liquid scintillationcounter (Perkin-Elmer, Wellesley, Mass.). Non-specifically bound (NSB)radioactivity was measured in the wells with 4 times greaterconcentration of “cold” native ligand than the highest concentration intest samples and total bound radioactivity was detected in the wellswith no competitor. Percent specific binding was calculated asfollowing: % Specific Binding=((Bound−NSB)/(Total bound−NSB))×100. IC₅₀values were determined by using Origin software (OriginLab, Northampton,Mass.).

Example 13

Functional Assay-cAMP Synthesis

The ability of glucagon analogs to induce cAMP was measured in a fireflyluciferase-based reporter assay. HEK293 cells co-transfected with eitherglucagon- or GLP-1 receptor and luciferase gene linked to cAMPresponsive element were serum deprived by culturing 16 h in DMEM(Invitrogen, Carlsbad, Calif.) supplemented with 0.25% Bovine GrowthSerum (HyClone, Logan, Utah) and then incubated with serial dilutions ofeither glucagon, GLP-1 or novel glucagon analogs for 5 h at 37° C., 5%CO₂ in 96 well poly-D-Lysine-coated “Biocoat” plates (BD Biosciences,San Jose, Calif.). At the end of the incubation 100 microliters ofLucLite luminescence substrate reagent (Perkin-Elmer, Wellesley, Mass.)were added to each well. The plate was shaken briefly, incubated 10 minin the dark and light output was measured on MicroBeta-1450 liquidscintillation counter (Perkin-Elmer, Wellesley, Mass.). Effective 50%concentrations were calculated by using Origin software (OriginLab,Northampton, Mass. Results are shown in Tables 2 and 3.

TABLE 2 cAMP Induction by Glucagon Analogs with C-Terminus ExtensioncAMP Induction Glucagon Receptor GLP-1 Receptor Peptide EC₅₀, nM N*EC₅₀, nM N Glucagon 0.22 ± 0.09 14 3.85 ± 1.64 10 GLP-1 2214.00 ±182.43  2 0.04 ± 0.01 14 Glucagon Cex 0.25 ± 0.15 6 2.75 ± 2.03 7Oxyntomodulin 3.25 ± 1.65 5 2.53 ± 1.74 5 Oxyntomodulin KRNR 2.77 ± 1.744 3.21 ± 0.49 2 Glucagon R12 0.41 ± 0.17 6 0.48 ± 0.11 5 Glucagon R12Cex 0.35 ± 0.23 10 1.25 ± 0.63 10 Glucagon R12 K20 0.84 ± 0.40 5 0.82 ±0.49 5 Glucagon R12 K24 1.00 ± 0.39 4 1.25 ± 0.97 5 Glucagon R12 K290.81 ± 0.49 5 0.41 ± 0.24 6 Glucagon Amide 0.26 ± 0.15 3 1.90 ± 0.35 2Oxyntomodulin C24 2.54 ± 0.63 2 5.27 ± 0.26 2 Oxyntomodulin C24 0.97 ±0.04 1 1.29 ± 0.11 1 PEG 20K *number of experiments

TABLE 3 cAMP Induction by Pegylated Glucagon Analogs cAMP InductionGlucagon Receptor GLP-1 Receptor Peptide EC₅₀, nM N* EC₅₀, nM N Glucagon0.33 ± 0.23 18 12.71 ± 3.74 2 Glucagon C17 PEG 5K 0.82 ± 0.15 4 55.86 ±1.13 2 Glucagon C21 PEG 5K 0.37 ± 0.16 6 11.52 ± 3.68 2 Glucagon C24 PEG5K 0.22 ± 0.10 12 13.65 ± 2.95 4 Glucagon C29 PEG 5K 0.96 ± 0.07 2 12.71± 3.74 2 Glucagon C24 PEG 20K 0.08 ± 0.05 3 Not determined Glucagon C24Dimer 0.10 ± 0.05 3 Not determined GLP-1 >1000  0.05 ± 0.02 4 *number ofexperiments

Example 14

Stability Assay for Glucagon Cys-maleimido PEG Analogs

Each glucagon analog was dissolved in water or PBS and an initial HPLCanalysis was conducted. After adjusting the pH (4, 5, 6, 7), the sampleswere incubated over a specified time period at 37° C. and re-analyzed byHPLC to determine the integrity of the peptide. The concentration of thespecific peptide of interest was determined and the percent remainingintact was calculated relative to the initial analysis. Results forGlucagon Cys²¹-maleimido PEG_(5K) are shown in FIGS. 1 and 2.

1. A derivative of native glucagon (SEQ ID NO: 1) comprising ahydrophilic moiety covalently bound to a residue at position 16, 17, or21, of the derivative, wherein the peptide stimulates activity at theglucagon receptor, and pharmaceutically acceptable salts of saidderivative.
 2. The derivative of claim 1 wherein said hydrophilic moietyis polyethylene glycol.
 3. The derivative of claim 2 wherein thepolyethylene glycol chain has a molecular weight selected from the rangeof about 1,000 to about 5,000 Daltons.
 4. The derivative of claim 2wherein the polyethylene glycol chain has a molecular weight greaterthan about 5,000 Daltons.
 5. The derivative of claim 4 wherein aminoacid 29 of the derivative is covalently bound to a second peptidecomprising a sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO: 20 and SEQ ID NO:
 21. 6. The derivative of claim 5,wherein the second peptide is SEQ ID NO: 19 and the terminal amino acidof the derivative comprises an amide group in place of the carboxylicacid group of the native amino acid.
 7. A multimer comprising at leasttwo derivatives of claim 1 bound to one another through a linker.
 8. Apharmaceutical composition comprising a derivative of claim 1, orpharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.
 9. A kit for administering a glucagon agonist to apatient in need thereof, said kit comprising a pharmaceuticalcomposition of claim 8; and a device for administering said compositionto a patient.
 10. A method of treating hypoglycemia in a patient, saidmethod comprising the steps of administering an effective amount of apharmaceutical composition of claim 8 to the patient.
 11. Thepharmaceutical composition of claim 8, further comprising insulin. 12.The derivative of claim 1, wherein the amino acid residue comprising thehydrophilic moiety is an amino acid selected from the group consistingof lysine, cysteine, ornithine, homocysteine, and acetyl phenylalanine.13. The derivative of claim 1, wherein the derivative comprises SEQ IDNO: 1 with an amino acid substitution at position 12 and/or position 27.14. The derivative of claim 1, wherein the derivative comprises an Argat position 12 and/or a Leu or Norleucine at position
 27. 15. Thederivative of claim 1, wherein the C-terminal amino acid of thederivative comprises an amide group in place of the carboxylic acidgroup of the native amino acid.